Mutagenesis, Vol. 14, No. 2, 243-248,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
A combined biochemical and cytogenetic study of thioridazine-induced damage to nucleic acids
1 Laboratory of Biochemistry, Faculty of Chemistry, Aristotle University of Thessaloniki 54006 and 2 Laboratory of Medical Biology and Genetics, The Medical School, Demokritus University of Thrace, PO Box 93, Alexandroupolis 68100, Greece
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In this work the biochemical effects of thioridazine, a commonly used phenothiazine, have been studied upon native double- and single-stranded DNA and also upon a supercoiled plasmid. The results indicate that thioridazine causes damage and scissions to these nucleic acids but only at concentrations much higher than the one used in our cytogenetic experiments and that the damage seems to depend on the concentrations used. Furthermore, we studied the action of thioridazine alone or in combination with caffeine and/or melphalan upon human lymphocytes in vitro. Thioridazine and caffeine (a well-known inhibitor of cellular repair mechanisms) were shown to act synergistically to potentiate the cytogenetic effect of melphalan on human lymphocytes. It is suggested that thioridazine alone or in combination with caffeine may exert its synergistic effect on melphalan cytotoxicity to cultured human lymphocytes not only indirectly, i.e. as a strong calmodulin inhibitor by facilitating the intracellular retention of melphalan, but also directly by reaction with nucleic acids and by causing scissions in and damage to them. Therefore, thioridazine (as chlorpromazine) has some potential as an adjuvant chemotherapeutic agent for the treatment of human cancer.
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Introduction |
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Certain phenothiazine tranquillizers, like chlorpromazine and thioridazine (TRZ), induce cytogenetic damage to normal human lymphocytes (Cohen et al., 1969
; Saxena and Ahuja, 1982; Lialiaris et al., 1988), are cytotoxic in different cell and tissue cultures (Boelsterli et al., 1987; Lehnert, 1987; Munyon et al., 1987; Strobl et al., 1990) and inhibit tumour growth in a variety of cell systems both in vitro and in vivo (Jones, 1985; Akiyama et al., 1986; Deen et al., 1986). Furthermore, phenothiazines are thought to be effective in improving the action of certain antineoplastic compounds, like melphalan (MEL) and others (Krishan et al., 1985; Lazo et al., 1986; Efferth and Volm, 1993), particularly in the presence of caffeine (CAF) (Cohen, 1975; Lialiaris et al., 1988, 1992). Similar findings, however, have not been demonstrated in all studies and there is some evidence showing that neither CAF (Ganapathi et al., 1984) nor TRZ (Carlo et al., 1986; Suryanarayana, 1991) have antineoplastic activity. These conflicting results, also reported by Gocke (1996), stimulated our study, which was set up to investigate: (i) the potential of TRZ, alone or in combination with CAF and/or MEL, to produce cytogenetic damage to cultured human lymphocytes; (ii) the ability of TRZ to cause damage to certain native nucleic acids.
The interactions and the possible mechanisms of binding of several antitumour drugs to DNA have been described (Waring, 1981). Many of them act as inhibitors of nucleic acid synthesis and interact with DNA by an intercalative, a non-intercalative or covalent mode of binding and, hence, usually cause conformational changes in the DNA. Unwinding of closed circular duplex DNA and lengthening of the helix and strand breakage are common and widespread responses to assault on DNA (Waring, 1981). TRZ may act in a similar way to these drugs.
SCEs have already been introduced as a very sensitive method for detecting mutagens and/or carcinogens in vitro (Tofilon et al., 1985; Deen et al., 1986; Lialiaris et al., 1990) and in vivo (Mourelatos et al., 1988; Lialiaris et al., 1992). Furthermore, other findings suggest that a common element, possibly a particular type of DNA damage produced by certain agents, is responsible for enhancing sister chromatid exchanges (SCEs) and for reducing cell proliferation and cell growth (Morris and Heflich, 1984; Lialiaris et al., 1987). SCE levels, proliferating rate indices (PRIs) and mitotic index (MI) values were, therefore, determined for the evaluation of cytogenetic effects, while double-stranded (ds) and single-stranded (ss) DNA isolated from thymus gland cells, and supercoiled plasmid DNA isolated from Escherichia coli were used as native nucleic acids.
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Materials and methods |
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SCEs in vitro
Heparinized blood samples were obtained from four healthy individuals, none of which was receiving drugs or was a smoker. Cultures of peripheral lymphocytes were prepared in universal containers by adding 11 drops of whole blood to 5 ml of chromosome medium 1A (Gibco). These were incubated at 37°C for 96 h. For SCE observations the cultures were first treated with thioridazine (0.5, 2.5 or 5 µg/ml) at 18 h and, 1–2 h later, with CAF (120 µg/ml) and/or MEL (150 ng/ml final concentration). Bromodeoxyuridine was then added at a concentration of 5 µg/ml. After 94 h a 0.3 µg/ml solution of colchicine was added and, at the end of the incubation period, cultures were harvested. Cultures were maintained in the dark to prevent or minimize photolysis of BrdU. Four separate and successful experiments took place, each experiment from each donor. In each experiment we used single cultures for seven treatments plus one control.
The chromosome preparations were stained by a modified fluorescence plus Giemsa technique (Goto et al., 1978). All chemicals were obtained from Sigma unless otherwise stated. Scoring was performed without prior knowledge of treatment. Cells were counted in their first, second or third and subsequent divisions. Mean SCE values were calculated only in suitable second division metaphases, because only in these can we observe and count the exchanges between the sister chromatids. To establish the PRI, in each culture 100 cells were counted and PRI calculated according to the formula PRI = (1M1 + 2M2 + 3M3+)/100, where M1 is the percentage of cells in the first division, M2 in the second and M3+ in the third and higher divisions. In addition MIs for 1000 activated lymphocytes were determined in each culture.
For the evaluation of synergy, the expected value (EV) was estimated from the formula EVMEL+CAF+TRZ = [OVMEL+CAF – OVMEL]+[OVMEL+TRZ – OVMEL] + OVME) , or better EVMEL+CAF+TRZ = OVMEL+CAF + OVMEL+TRZ – OVMEL, where EV is the expected value and OV is the observed value of treatments.
Statistical analysis
Usually according to previous as well as present evaluations of our results, SCE frequencies among individual cells don't follow a normal distribution and, therefore, use of standard parametric statistical methods is contraindicated. The results need to be transformed in a logarithmic or square root way. So, to compare various treatments, logarithmic transformation of SCEs was first performed using the one-way analysis of variance (ANOVA) and subsequently the Dunkan test for the calculations concerning pair-wise comparisons. It is assumed that culture-to-culture variation is small compared with cell-to-cell variability so that the cell is used here as the experimental unit for statistical purposes.
Evaluation of MI and PRI was based on the 
2 test. Furthermore, correlations between SCEs and MIs or SCEs and PRIs were calculated as reported by Ioannidou et al. (1989) and Lialiaris et al. (1989). Finally, a t-test was performed to evaluate the synergy between expected value (EV) and observed value (OV).
Nucleic acids
Plasmid pKS (Bluescript) was isolated from E.coli by the alkaline SDS lysis method (Promega Biotech). Native DNA was isolated from thymus gland cells using a standard procedure and denatured ssDNA was prepared by heating it at 100°C for 10 min. Linear DNA was prepared after incubation of plasmid pKS by the restriction enzyme BamHI. All plastics and glassware used in the experiments employing nucleic acids digests were autoclaved for 30 min at 120°C and 130 kPa. Heat-resistant solutions were similarly treated, while heat-sensitive reagents were dissolved in autoclaved water.
Assay conditions
Aliquots of 1–3 µg of each nucleic acid (as indicated in the legends) were incubated in the presence of thioridazine at different concentrations in a final volume of 15 µl. TRZ was in the form of the HCl salt and all solutions were neutral. The reaction was for 30 min at a constant temperature of 37°C. It was terminated by the addition of 5 µl loading buffer consisting of 45% Ficoll (type 400), 1% bromophenol blue, 1% xylene cyanol in water. The products resulting from TRZ interactions were separated by electrophoresis on 1% agarose gels, which contained 1 µg/ml ethidium bromide in 40 mM Tris–acetate, pH 7.5, 20 mM sodium acetate, 2 mM Na2EDTA at 5 V/cm. Agarose gel electrophoresis was performed with a horizontal gel apparatus (Mini-Sub(TM) DNA Cell; BioRad) for ~4 h. Since ethidium bromide forms a fluorescent complex when it binds to DNA, a decreased fluorescence signifies diminution of the amount of DNA. The gels were visualized in the presence of UV light. All assays were duplicated (Lialiaris et al., 1992).
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In Figures 1–3
the effects of TRZ on the integrity of the nucleic acids are shown. Concerning the pDNA, local unwinding is evidenced by monitoring the effects of TRZ binding on the superhelical state of circular DNA; the interaction of TRZ with the pDNA was obvious starting from the lowest concentration used (0.5 mM) to the highest (5 mM). This was concluded from the disappearance of the supercoiled (form I), an alteration in the amount of the relaxed (form II) and the appearance of formations (or forms III) of higher molecular weight, which are characterized in similar cases as knots or catenanes. These forms perhaps resulted from a cross-linking between the TRZ molecules and the plasmid. In order to identify the mobility of the linearized plasmid in the agarose gel, the same plasmid pKS was digested with the restriction enzyme BamHI and electrophoresed under the conditions described in Figure 3A.
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DNA HindIII fragment markers, 23 130 , 9416, 6557, 4361, 2322, 2027, 564 and 125 bp.
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Concerning the ssDNA and dsDNA, there was remarkable damage amounting to catabolism of the dsDNA and ssDNA, with possible scissions, fragmentation and partial to complete disappearance of nucleic acid bands. Further, TRZ was effective at concentrations >1 mM for ssDNA and 2 mM for dsDNA and the damage produced was dose dependent (Figures 1–3
). Autoclaved plastics, glassware and solutions were used to avoid various nuclease activities. This can be seen by the absence of any activity on the controls used.
The potential of TRZ to induce cytogenetic effects on cultured human lymphocytes, alone or in combination with CAF and/or MEL is shown in Tables I–V. There were four separate and successful experiments, each of which employed a different donor. In each experiment we used eight single cultures, one for each of the possible combinations. The data for each experiment are shown in Tables I–IV and the last two combined in Table V (as we used the same concentration of TRZ in these). So, for each culture we evaluated 25–35 metaphases for SCE evaluation. For this reason, small SEMs were expected as a result of the large number of metaphases. In our experiments we used 0.5, 2.5 and 5 µg/ml TRZ because we found that at concentrations >10–20 µg/ml TRZ in vitro induced strong cytotoxicity to human lymphocytes. As a consequence, we used lower concentrations of TRZ to avoid toxicity and to ensure better readable results for all combinations.
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Thus, TRZ alone at a final concentration of 0.5–5 µg/ml could not induce any cytogenetic damage, but in combination with CAF enhanced (P < 0.05) SCE levels (Tables III–V
). CAF alone caused a small statistically insignificant increase in SCE, while MEL alone produced significant (P < 0.01) induction of SCE frequency. Small changes in PRI and MI with these treatments were not statistically significant. Furthermore, TRZ at 5 µg/ml caused cytogenetic damage: (i) in combination with caffeine (CAF) or with melphalan (MEL), enhancing SCE levels and producing cell division delays and suppression of MIs; (ii) in the triple combination with CAF and MEL, as can be deduced from the observed synergism on induction of SCE levels (P < 0.01), in suppressing (P < 0.05) PRI and MI values. According to the references, MEL induces a high SCE frequency but doesn't produce strong cytotoxicity. So, it seems that MI and, of course, PRI values are constant with the exception of culture 8, which was treated with the triple combination MEL+CAF+TRZ (Tables III–V). Only the triple combination (MEL+CAF+TRZ) showed synergistic action on cultured human lymphocytes. This derives from the evaluation of the expected SCE value (EV = 53.73) compared with the observed value (OV = 68.12 in Table V). Irrespective of whether expected values (EV) were calculated using the double combinations with MEL (e.g. EV of [MEL] + [TRZ+CAF], which is equal to 54,25) or the single combinations (EVs ranged from 53.73 to 59.25), synergism was demonstrable (P < 0.05) for MEL+CAF+TRZ since the OV (68.12) was well above the EV. We preferred to present the EV concerned directly with the effect of MEL in the tables.
Finally, there was a negative correlation between SCE enhancement and reduction in PRIs (r = –0.756, P < 0.05) or suppression of MIs (r = –0.882, P < 0.01) and a positive correlation between PRI reduction and MI suppression (r = +0.775, P < 0.05) in Table V.
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TRZ acting directly on DNA could cause scissions, fragmentation and disappearance of nucleic acid bands, though admittedly at somewhat high concentrations (0.5–5 mM, 0.2–2.0 mg/ml) in vitro for a limited time (30 min). On the other hand, the concentrations used in the cytogenetic experiments were comparatively low (0.5–5 µg/ml) and the time of incubation was much longer (96 h). The causes of these effects have not been demonstrated. Various possibilities are consistent with our data, such as formation of free radicals (Vazquez et al., 1992
) or action as an intercalating agent in dsDNA, like other phenothiazines (Darkin et al, 1984; Lialiaris et al., 1992) or binding covalently to DNA (Schoonderwoerd et al., 1990) or by interference with various enzymatic reactions (Boelsterli et al., 1987) and, perhaps most importantly, by interference with cellular membranes as a potent calmodulin inhibitor (Jones, 1985; Lehnert, 1987; Strobl et al., 1990). In this paper we report cleavage of all the DNA substrates with increasing concentrations of TRZ (Figures 1–3) but the exact mechanism of TRZ action is not clearly understood.
To examine the involvement of DNA secondary structures and the action of TRZ on them, we assayed dsDNA and supercoiled DNA. The cleavage of supercoiled DNA could be of biological significance for the reason that native DNA is negatively supercoiled. TRZ appears to catalyse non-specific cleavage of the plasmid (Figures 2 and 3B).
Furthermore, we tested the biological effect of TRZ alone or in combination with CAF and/or MEL using the SCE methodology (Perry and Evans, 1975). Studies to search for a relationship between SCE induction and other expressions of genotoxicity have shown a positive relationship between SCEs and reduced cell survival and alterations in cell cycle kinetics (Morris and Heflich, 1984; Lialiaris et al, 1988, 1989; Maskaleris et al., 1998). Similar results were obtained in our paper (Table V). The combined cytogenetic and biochemical findings of this study indicate that TRZ alone, but mainly in combination with CAF and/or MEL, causes serious damage to DNA. Only the triple combination (MEL+CAF+TRZ) showed synergistic action on cultured human lymphocytes in terms of the SCE, PRI and MI values. Results similar to these have been presented in a previous work (Lialiaris et al., 1992).
It seems that successful DNA repair, prior to S phase, removes damage that might otherwise give rise to SCEs (Faed and Mourelatos, 1978). Indeed, TRZ combined with CAF tends to increase the activity of the antineoplastic agent MEL (Tables III–V) and this effect is thought to be related mainly to the inhibitory action that CAF exerts on certain enzyme repair mechanisms (Lialiaris et al., 1988). Our results are in line with most studies (Cohen et al., 1967; Cohen, 1975; Ganapathi et al., 1984; Lialiaris et al., 1988) although in a few a damaging effect of TRZ has been not demonstrated (Carlo et al., 1986; Suryanarayana, 1991). Of course, the results of such experiments can only be transferred to man by inference. However, in certain studies it was found that TRZ caused cytotoxicity in a variety of cells and tissues (Boelsterli et al., 1987; Lehnert, 1987; Munyon et al., 1987; Strobl et al., 1990; Nankivell et al., 1994; Buckley et al., 1995) and this may be of importance in prevention of mutagenicity of TRZ in patients with neuroleptic treatment.
It has been shown that the determination of PRIs and MIs should be a very sensitive and useful indicator of cytotoxicity in human lymphocyte cultures due to DNA damaging agents (Snope and Rary, 1979; Lialiaris et al., 1989, 1992; Maskaleris et al., 1998). The highest SCE rates appeared in cultures demonstrating the greater suppression of PRIs and/or MIs. In our work a sound correlation between SCE induction and cell division delay and/or MI was observed (Table V).
If TRZ and/or CAF do interfere with DNA repair in man, as seems likely, it should be possible to increase the effectiveness of alkylating agents by inhibiting or deranging the DNA repair mechanism. The concentration of TRZ used (0.5–5 µg/ml) is below the toxic levels in blood (toxic at >10 µg/ml), so low and non-toxic concentrations of an intercalating agent (like thioridazine) could be used in combination with different alkylators for better antineoplastic results.
Finally, as mentioned before, we found that chromosomes treated with TRZ are more sensitive to MEL and CAF compared with chromosomes with no previous TRZ treatment. We therefore propose that clinical use of TRZ in combination with antitumour agents may be of interest in the treatment of human cancer.
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3 To whom correspondence should be addressed. Tel: +30 551 39888; Fax: +30 551 39898; Email: lialiari{at}demokritos.cc.duth.gr
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Received on October 6, 1998; accepted on December 1, 1998.
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